PAP, the antiviral protein extracted from pokeweed plants, is known to retard virus transmission by enzymatically inhibiting protein synthesis on eucaryotic ribosomes. The mode of inhibition is similar to that of the known antitumor enzyme ricin. We have crystallized PAP (and also ricin) in a form suitable for x-ray diffraction analysis. During the first two years of our initial grant, we developed a system to process high resolution x-ray data collected by oscillation photography. We have collected 2.5A data for PAP and three derivatives, IrCl3, PtCl4 and ICl, and are now interpreting this data as part of phase determination. In addition to our x-ray studies we have determined that PAP requires a "helper" protein from the postribosomal supernatant and either ATP or GTP for maximum inhibition of ribosomes. When these added factors are present, PAP shows a Kcat equals 500 min-1 and Km equals 0.4 micron m for ribosomes. We also found that arginine residues are required for PAP activity, indicating the enzyme may recognize the rRNA backbone. We propose to continue this investigation. Specifically we propose to produce a 2.5A election density map for PAP, to build a 2 cm/A model of the protein and to carry out a least squares refinement of that structure using semiautomated techniques. We propose to analyze the substrate binding site by soaking substrate analogs into the crystals or, failing that, to build theoretical models of nucleotide interactions. A comparison will also be made with the structure of ricin. Finally, we propose to extend our chemical and kinetic studies to include 1) an isolation and characterization of the "helper" protein and its role in the toxin activity, 2) the role of ATP and GTP in the mechanism, 3) kinetic studies on partially inhibited PAP and binding studies to find substrate analogs (probably oligonucleotides) which will bind to the enzyme. We hope the structural and chemical studies proposed here will prove to be useful in drug design.